Epstein Barr Virus (EBV) is causally related to Burkitt's Lymphoma, Hodgkin's Lymphoma, and Lymphoproliferative Diseases in immune suppressed and HIV infected people. In humans deficient in T cell immune responses, B-cells expressing the EBV Latency III growth program can be highly malignant. EBV conversion of resting B lymphocytes to continuously proliferating lymphoblastoid cells (LCLs) is a relevant and experimentally useful model for EBV oncogenesis. The Latency III EBV nuclear antigens EBNA1, 2, LP, 3A, 3C, and the membrane protein LMP1 are required for EBV B-cell transformation. In LCLs, EBNA2 is the principal EBV nuclear activator of cell and virus gene expression, and exerts its effects through the cell transcription factor RBPJ. EBNA2 binds to mostly non-promoter sites and is likely to use chromatin looping to regulate gene expression from distal sites. The EBNA3s compete with EBNA2 for RBPJ, potentially down-modulating EBNA2 RBPJ-dependent effects. Although EBNA3A, EBNA3B, and EBNA3C are highly similar, they differentially affect cell gene expression and only EBNA3A and EBNA3C are essential for LCL growth. Reverse genetic studies indicate that EBNA3A and EBNA3C interaction with RBPJ are both critical for LCL growth. EBNA3A and 3C suppress CDKN2A/B locus p16 and p14 expression to enable continuous cell proliferation. siRNAs to p16 and p14 enable cell proliferation in place of EBNA3A or 3C. To further characterize the molecular mechanisms through which EBNA2 and EBNALP regulate transcription, our specific aims are to: 1. Determine the molecular mechanisms by which EBNA3A, EBNA3C, or EBNA3B (as a control) affect gene expression through genome-wide interactions alone or with RBPJ and alter EBNA2 interactions with RBPJ and DNA, using ChIP-seq. 2. Determine the mechanisms through which EBNA3A and EBNA3C suppress p16, p14, (and p15) expression using conditional EBNA3A and 3C LCLs and time course studies of RBL infection. 3. Determine the basis for the EBNA3C N-terminal and C-terminal domains uniquely critical for LCL growth. These studies will provide new insights on how EBNA3A and EBNA3C contribute to EBV mediated growth transformation. PUBLIC HEALTH RELEVANCE: Epstein Barr Virus (EBV) associated malignancies are a significant threat to humans. These studies will elucidate the molecular basis through which EBV proteins EBNA3A and EBNA3C exploit the normal B lymphocyte transcription program to achieve immortal growth and identify potential targets for intervention.